Therapeutic treatment of cancer and dysplasia of the cervix or vagina using estrogen antagonists

ABSTRACT

A method for treatment of cervical or vaginal cancer and their associated dysplasia, including the steps of identifying a human cervical or vaginal cancer and/or dysplasia patient, administering an effective amount of an estrogen antagonist therapy to the patient, wherein the amount is effective to reduce cancer and dysplasia symptoms, and observing a reduction of cancer and dysplasia symptoms in the patient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent Ser. No. 12/693,850,filed Jan. 26, 2010, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/147,667, filed Jan. 27, 2009, U.S. ProvisionalApplication Ser. No. 61/148,445, filed Jan. 30, 2009, and U.S.Provisional Application Ser. No. 61/261,615, filed Nov. 16, 2009. Eachof these applications is incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe following agency: NIH CA120847. The United States government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

High-risk human papillomavirus (HPV) types, particularly HPV16, arecausally associated with human malignancies including cervical andvaginal cancers in women (zur Hausen H, 2002). New prophylactic HPVvaccines can prevent infections by a subset of these HPVs; however, theywill not eliminate pre-existing HPV infections, new infections byhigh-risk HPVs not targeted by the vaccines, or cervical cancers andprecancerous lesions that arise from those HPV infections (Roden and Wu,2006). Traditional therapeutic approaches (for example, surgery,radiotherapy, and chemotherapy) are of limited value to patients withadvanced or recurrent cervical cancer. Consequently, cervical cancerremains the second leading cause of death by cancer among womenworldwide (zur Hausen H, 2002; Roden and Wu, 2006). New, more effectivetherapeutic strategies are clearly needed. Herein, a potent newtherapeutic approach that not only effectively treats preexistingcervical and vaginal cancers but also can prevent their onset isidentified.

The uterine cervix is highly responsive to steroidal hormones such asestrogen. Correspondingly, cervical cancers most commonly arise in the3rd-5th decades, which typically correspond to the pre-menopausal periodof life in women (Cline, 2004). Furthermore, use of oral contraceptivesor high parity has been shown to significantly increase the risk forcervical cancer in HPV-infected women (Moreno et al., 2002; Munoz N etal., 2002). These observations raise the possibility that steroidalhormones such as estrogen might affect cancers of the cervix, much likethat of other hormonally responsive female organs (Cline, 2004; Jordan,2007). Estrogen replacement therapy alone, however, does not increasethe risk for cervical cancer, and tamoxifen, a well-known estrogenreceptor antagonist in the breast, has no beneficial effect on cervicalcancer (Bigler et al., 2004; Persson et al., 1996). Unfortunately,neither of these studies controlled for infections with high-risk HPVsthat are prerequisite for cervical cancer (zur Hausen H, 2002; Roden andWu, 2006). Furthermore, tamoxifen has an estrogen receptor (ER)agonistic rather than antagonistic effect in the human cervix (Friedrichet al., 1998). Studies have shown a protective effect on cervical cancerby indole-3-carbinol, a compound found in cruciferous vegetables thatfavorably alters the metabolism of estrogen; however, this drug has alsobeen shown to inhibit the genesis of other tumors that are not believedto depend upon estrogen (Aggarwal and Ichikawa, 2005; Jin et al., 1999;Bell et al., 2000). Thus, the evidence for or against a role of estrogenin cervical carcinogenesis in humans has been limited in nature andinconclusive.

To elucidate mechanisms by which HPV oncogenes promote cervical cancerand by which cofactors contribute to cervical carcinogenesis, we havegenerated transgenic mouse models that express HPV16 E6 and/or E7 underthe control of human keratin 14 promoter that drives gene expression instratified squamous epithelia, natural targets for HPV infection. Theprogressive disease that arises in these mouse models recapitulatesvarious aspects of human cervical disease, including the multiple stagesof cervical carcinogenesis, the anatomical location andhistopathological nature of the cancers, and the expression patterns ofvarious biomarkers (Brake et al., 2003; Elson et al., 2000), validatingthe relevance of these preclinical models to cervical disease in women.Using these mouse models, we have demonstrated that estrogen is requiredfor development of cervical cancer and that estrogen receptor alpha(ERα) is crucial for the development of atypical squamous metaplasia(ASM), which has been proposed to be the very first step of cervicalcarcinogenesis preceding the development of cervical intraepithelialneoplasia (CIN), a precancerous cervical lesion that can progress tocervical cancer (Elson et al., 2000; Brake and Lambert, 2005; Chung etal., 2008; Shai et al., 2007; Arbeit et al., 1996; Riley R R, et al.2003). It is still unclear, however, whether ER-α is crucial for theprogression of CIN lesions to invasive cancer and maintenance ofcervical cancer. Herein, it is demonstrated that inhibition of ER iseffective in both treating and preventing cervical cancer in mice.

SUMMARY OF THE INVENTION

In one aspect according to the present invention, a method for treatmentof cervical or vaginal cancer or their associated dysplasia (CINI-III,VINI-III)) is disclosed. The method includes the steps of identifying ahuman cervical or vaginal cancer and/or dysplasia (CIN/VIN) patient andadministering an effective amount of an estrogen antagonist therapy tothe patient. The amount is effective to reduce cancer and dysplasiasymptoms. The method further includes observing a reduction of cancerand dysplasia symptoms in the patient.

In a second aspect according to the present invention, a method forpreventing cervical or vaginal cancer in patients with dysplasia isdisclosed. The method includes the steps identifying a dysplasia(CIN/VIN) patient and administering an effective amount of an estrogenantagonist therapy to the patient. The amount is effective to reducedysplasia symptoms and prevent cervical cancer. The method furtherincludes measuring a reduction of dysplasia symptoms and absence orslowing of progression to cervical/vaginal cancer.

In a third aspect according to the present invention, a method forpreventing recurrence of cervical or vaginal cancers is disclosed. Themethod includes the steps of identifying a human cervical or vaginalcancer patient who has been successfully treated to remove detectablecancer, administering an effective amount of an estrogen antagonisttherapy to the patient, and monitoring for persistence of a disease-freestate.

In a fourth aspect according to the present invention, a method oftreating ERα-dependent spreading or metastatic cancers of cervical orvaginal tissue origin is disclosed. The method includes identifying ahuman cancer patient with spreading or metastatic cancer of cervical orvaginal tissue origin, administering an effective amount of an estrogenantagonist therapy to the patient, and measuring reduction of cancersymptoms in the patient.

DESCRIPTION OF THE DRAWINGS

FIG. 1. One-month fulvestrant treatment results in regression ofcervical cancer as well as dysplasia. FIG. 1A shows low magnificationimages of representative H&E-stained endocervical sections from eachgroup of mice described in Tables 1 and 2. FIG. 1B shows highmagnification images of representative H&E-stained lower reproductivetracts. In both FIGS. 1A and 1B CIN2/3 lesions as well as cancers areevident in mice not treated with fulvestrant (left two panels) but areabsent in mice treated with fulvestrant (right two panels). Note alsothat dysplasia is absent in mice treated for only 2 weeks withfulvestrant but cancers remain.

FIG. 2. Cancers remaining after one-month fulvestrant treatment areassociated with non-hypoplastic epithelium. Shown in the left panels arelow magnification images of the two cancers (one cervical—upper panel,the other vaginal—lower panel) that persisted in 2 of 13 mice treatedwith fulvestrant for one month. In the right panels are highmagnification images of cervical (upper panel) or vaginal (lower panel)epithelium that was immediately juxtaposed to these two cancers. Notethe non-hypoplastic (i.e., thick) epithelium akin to what is seen infemale mice in normal estrus. This is in stark contrast to thehypoplastic epithelia found elsewhere in the reproductive tracts ofthese same fulvestrant-treated mice (see inset images in right panels),as well as in the other fulvestrant treated mice (see FIG. 1B).

FIG. 3. Screening for ERα-positive human cervical cancer cell lines.Various cervical cell lines were examined for ERα expression byimmunoblotting with an anti-ERα antibody (H184 from Santa CruzBiotechnology, Inc., Santa Cruz, Calif.). The cell lines screenedinclude: 293, an ERα⁻ epithelial cell line (human; negative control);MCF7, an ERα⁺ breast cancer cell line (positive control); CasKi; SiHa;ME180; Hela (purchased from ATCC; CVX1 (Bellone et al., 2003), an HPV⁺cervical cancer cell line; C33A, an HPV⁻ cervical cancer cell line; andCIN612E (Bedell et al., 1991), an HPV⁺ cervical dysplasia cell line.Actin was used as a loading control.

FIG. 4. Formation of tumors in nude mice subcutaneously injected withCVX1 human cervical cancer cells. CVX1 cells were subcutaneouslyinjected into nude mice that were either untreated (−E2) orestrogen-treated (+E2). Tumor formation was monitored, and tumor-freestate was plotted with a Kaplan Meier curve.

FIG. 5. Effectiveness of fulvestrant in blocking growth in ERα-positivevs. ERα-negative cervical cancer cell lines. Estrogen-treated nude micewere injected with CVX1 (a) or CaSki (b) cells. Once tumors formed, onegroup of mice was treated with fulvestrant twice a week and the othergroup was left untreated. Tumor growth was monitored every week (a) orevery 4 days (b). Tumor size was determined by the diameter of crosssectional area and starting size was set at 100%. CVX1 tumor size at thetime of fulvestrant treatment was 7.7±2.4 mm and that of the untreatedgroup was 3.9±1.5 mm. CaSki tumor size in both groups was 3 mm onaverage.

DESCRIPTION OF THE INVENTION A. Introduction

Despite the recent advent of human papillomavirus (HPV) vaccines,HPV-associated cancers of the lower female reproductive tract, cervicalcancer being most common among these, remain a great health burdenworldwide and are predicted to remain so for at least the next threedecades. Cervical cancer alone is the second most common cancer in womenand the second most frequent cause of death by cancer in women worldwide(Sankaranarayanan and Ferlay, 2006). Current therapeutic approaches fortreating patients with cervical cancer, primarily radio- andchemo-therapies, have limited success, with 5-year survival rates beingless than 50% worldwide (Sankaranarayanan and Ferlay, 2006).

Cervical cancers arise through multiple steps, cervical intraepithelialneoplasia 1 (CIN1, low-grade dysplasia), CIN2 (mid-grade dysplasia),CIN3 (high-grade dysplasia), and then finally invasive cancer. AlthoughCIN lesions may regress spontaneously, CIN3 is normally treated due toits high rate of progression to cancer. Treatment of cervical dysplasiastraditionally involves surgical procedures such as Loop ElectrosurgicalExcision Procedure (LEEP) and cryo/laser therapies, all of which areassociated with complications (Hunter et al., 2008a; Hunter et al.,2008b).

Virtually all cervical cancers are associated with the so-calledhigh-risk human papillomaviruses (HPVs), among which HPV-16 is mostcommon, being found in approximately 60% of all cervical cancers (Burd,2003). A newly available vaccine that inhibits infection by a subset ofcancer-causing HPVs including HPV-16 shows promise for reducing cervicalcancer in future generations of women who make use of this vaccine(FUTURE II Group, 2007; Villa, 2007). It is clear, however, that moreeffective strategies are warranted for current generations of women whoare already exposed to high-risk HPVs and for future generations ofwomen who are not vaccinated or become infected with cancer-causing HPVsnot targeted by currently available vaccines. Furthermore, a specificnon-invasive therapy for cervical cancer and/or dysplasia is currentlyunavailable, as described above.

Although it is well established that HPV is the major etiological factorfor cervical carcinogenesis, HPV infection alone is not sufficient forthe development of cervical cancer because only a minor fraction ofpatients infected with HPV develop cervical cancer and CIN lesions oftenregress without treatment (Schiffman et al., 2007). Several cofactorsincluding long-term use of oral contraceptives and high parity have beenimplicated in the genesis of HPV-associated cervical cancer (Moreno etal., 2002; Munoz et al., 2002). These results raise a possibility that,in addition to HPV, female steroid hormones such as estrogen may play acrucial role in cervical carcinogenesis similar to breast cancers(Palmieri et al., 2002).

It is relevant that infertile women using an anti-estrogen, clomiphenecitrate, have reduced cervical cancer incidence compared to non-users(Rossing et al., 1996). This study, however, did not control for HPVinfection that is prerequisite for the development of cervical cancer(Woodman et al., 2007; zur Hausen, 2002). In addition, it is difficultto reconcile conflicting observations that use of clomiphene citrateincreases risk for uterine and breast cancers (Althuis et al., 2005a;Lerner-Geva et al., 2006). Furthermore, another study showed thatinfertile women using clomiphene citrate have slightly higher risk forcervical cancer, though statistically not significant (Althuis et al.,2005b).

Other studies argue that cervical cancers do not rely upon estrogen, butthose studies also have caveats. For instance, one observational studywithout controlling for HPV infection argues that hormone replacementtherapy with estrogen alone or estrogen-progestin combination does notincrease risk of cervical cancer (Persson et al., 1996). Anotherclinical study demonstrates that a selective estrogen receptor modulator(SERM) tamoxifen has no beneficial effect on cervical cancer, leading tothe conclusion that cervical cancer is estrogen-independent (Bigler etal., 2004). An assumption of this study was that tamoxifen is anantagonist for estrogen receptor (ER) in the cervix as in the breast. Ithas been shown, however, that tamoxifen has an agonistic rather thanantagonistic effect on ER function in the human cervix (Friedrich etal., 1998). Furthermore, it is difficult to interpret the relevance ofconfounding responses of different cervical cancer cell lines toestrogen and various SERMs because these cells have been adapted togrowth in tissue culture for decades, and therefore, may not beadequately representational of in vivo conditions, and expressionpatterns of ER are poorly characterized (Herynk and Fuqua, 2004). Thus,there remains a poor understanding of the estrogen-dependence ofcervical cancers in humans.

An essential role of estrogen in cervical cancer has been clearlydefined in mouse models for HPV-associated cervical cancer that make useof transgenic mice expressing major HPV oncogenes E6 or E7, or bothunder the control of human keratin 14 (K14) promoter, which drivestransgene expression in stratified squamous epithelia, natural HPVinfection sites. In these mouse models, either an HPV oncogene orestrogen alone is insufficient to cause cervical cancers, whereas an HPVoncogene in conjunction with physiological levels of exogenous estrogencan promote the development of cervical cancer (Brake and Lambert, 2005;Elson et al., 2000; Riley et al., 2003; Shai et al., 2007). Theprogressive cervical disease that arises in these mouse modelsrecapitulates various aspects of human cervical disease, including themultiple stages of cervical carcinogenesis (atypical squamousmetaplasia, CIN 1-3, and then cancer), anatomical location (cancersprimarily arising from the transformation zone), histopathologicalnature of the cancers, and the expression patterns of various biomarkers(Brake et al., 2003; Elson et al., 2000).

It is hypothesized that estrogen can contribute to the development ofcancers by ER-dependent and -independent mechanisms. Estrogen is bestknown for exerting its physiological effects by binding and activatingits receptors, ERα and/or ERβ (Couse and Korach, 1999). The mitogeniceffects of estrogen that are mediated through ERα are crucial for thedevelopment and maintenance of most human breast cancers (Palmieri etal., 2002). ERα-positive breast cancers are highly responsive to thetherapy with SERMs such as tamoxifen and fulvestrant that directly bindand inhibit the function for ERα (Heldring et al., 2007). The functionfor ERβ in breast cancer and other estrogen-dependent cancers is lesswell understood. The potential ER-independent mechanism involvesestrogen metabolites that can function as a direct carcinogen inducingdetrimental genetic mutations (Cavalieri et al., 2006). However, itremains unclear to what extent this mechanism contributes to estrogen'srole in estrogen-dependent cancers.

With regard to the mechanism by which estrogen cooperates with HPVoncogenes in murine cervical carcinogenesis, we have recently obtainedevidence that estrogen functions through ERα, as ERα null miceexpressing E7 (K14E7/ERα^(−/−)) lack cervical cancers as well as CINeven after treatment with estrogen for 6 months. It is still unclear,however, whether ERα is crucial for the progression to and maintenanceof cervical cancer and CIN lesions because K14E7/ERα^(−/−) mice treatedwith estrogen fail to develop atypical squamous metaplasia that has beenproposed to be the very first step of cervical carcinogenesis precedingthe development of CIN (Chung et al., 2008).

In the Examples presented below, we examined whether anti-estrogens havea beneficial effect on treating and preventing cervical cancers arisingin K14E6/K14E7 double transgenic mice treated with estrogen for 6months, as well as on treating tumor formation in a human cervicalcancer model promotes regression and prevention of cervical and vaginaldisease including cancer. Our second example demonstrates thatraloxifene, a selective estrogen receptor modulator (SERM), alsopromotes regression of cervical and vaginal disease including cancer.Our third example demonstrates that fulvestrant prevents the developmentof cervical cancer. The fourth example demonstrates that estrogenfacilitates tumor formation by an ERα-positive human cervical cancercell line (CVX1) in nude mice and that fulvestrant inhibits tumor growthby this cell line.

B. Method of Treating and Preventing Cervical and Vaginal Cancers

In one embodiment, the present invention is a method of treatingcervical or vaginal cancer and dysplasia comprising the administrationof an anti-estrogen to a cervical or vaginal cancer and/or cervical orvaginal dysplasia patient. In a preferred embodiment, one would (1)obtain a diagnosis of cancer or dysplasia in a patient, (2) administeran effective amount of an estrogen antagonist, wherein the amount iseffective to reduce cancer or dysplasia symptoms, and (3) examine thepatient for reduction of cancer symptoms. Section (C) below disclosespreferred anti-estrogen therapies.

One would obtain a diagnosis of cervical/vaginal cancer or dysplasiathrough conventional means. For example, one may refer to Jhingran etal., Churchill Livingstone Elsevier Press, Philadelphia (2008), pp1745-1792.

The paragraphs below describe a preferred prophetic treatment of a humancervical cancer and dysplasia patient:

We have found that estrogen antagonist therapies, such as fulvestrantand raloxifene, have multiple applications in the treatment of squamouscell carcinoma of the uterine cervix or vagina. These applications couldbe applied in both pre-menopausal and post-menopausal women. Estrogenantagonist therapy could be considered as primary single agent therapyor be used in combination with other classic chemotherapy agents, suchas platinum and taxane molecules, as anti-estrogen therapy may enhancethe cytotoxic effects of these drugs (despite the fact that squamouscell carcinomas are historically felt to be largely resistant to suchmedications). Clearly the most straightforward application would be astherapy in pre-menopausal and peri-menopausal women wherein estrogen,arising systemically or from the lesion itself, is a driving factor intumor development, maintenance and/or progression.

In general, eligible patients for this type of treatment would have adiagnosis of cervical/vaginal cancer with a documented occurrence orrecurrence of disease that is measurable by clinical exam or radiologicstudies (such as CT scans). Once identified as subjects, women will betreated with estrogen antagonist therapy per established dosing regimens(derived primarily from breast cancer treatment). If fulvestrant is theselected anti-estrogen, preferable treatment would include loading dosesof 500 milligrams on day 1 and 14 and then monthly dosing at one of twostandard doses, 250 mg or 500 mg every 28 days (Robertson J F.,Oncologist. 2007 12:774-84) delivered IM. One may wish to optimize thedosage for cervical cancer. Doses of between 50-1000 mg would beconsidered useful. Treatment with the anti-estrogen could be done aloneor in conjunction with existing chemotherapeutic modalities.

Preferably, patients will be seen every 28 days and tumor measurementsfor response checked every other month. Responses will be defined bystandard RECIST (Therasse P, et al. J Nat Cancer Inst 2000; 92:205-16)criteria and response rates compared to those of other drugs used totreat this disease in the past. Potentially favorable response rateswould be an indication to continue with optimal development in thisdisease. Due to the diverse hormonal status of eligible women, atreatment such as this will need to be evaluated based on such hormonalfactors as menopausal status, body mass (obesity), as well as receptorstatus within the tumor itself and/or associated stroma. Each of thesefactors could influence the optimum choice and dose of anti-estrogen andthe predicted responsiveness to the treatment.

As described above, in one embodiment, one would treat the patient ofthe present invention with fulvestrant in a manner similar to treatmentof breast cancers with fulvestrant. If one elected to use otheranti-estrogen therapies, e.g., raloxifene, a dose similar to that inapproved use would be preferred. For example, if one wished to useraloxifene, an oral dose of between 6 and 600 mg (±10%) a day,preferably 60 mg a day, would be preferred.

In another embodiment, patients who had been treated forcervical/vaginal cancer and/or precancerous lesions would be placed onanti-estrogen therapy to prevent recurrence of disease. Such patientswould be dosed as described above akin to the treatment of breast cancerpatients for whom anti-estrogen therapy has proven effective atprolonging disease free state and survival.

In another embodiment, the present disclosure provides a method oftreating ERα-dependent spreading or metastatic cancers of cervical orvaginal tissue origin. The method includes: (1) identifying a humancancer patient with metastatic or spreading cancer of cervical orvaginal tissue origin; (2) administering an effective amount of anestrogen antagonist therapy; and (3) observing reduction of cancersymptoms in the patient. Observation may take the form of measuring thereduction of cancer symptoms in the patient.

While not wishing to be bound by theory, it is believed that cancers ofcervical and/or vaginal tissue origin prior to becoming and/or at thesame time as they become metastatic, may spread from the cervix and/orvagina into immediately surrounding tissues, including for example,those associated with the peritoneum. As well, it is possible that atsome point during the progression of cervical and/or vaginal cancers,metastasis will occur resulting in the spread of cervical and/or vaginalcancer cells throughout the body. To the extent that these spreadingand/or metastasizing cervical and vaginal cancers are ERα-dependent, anestrogen antagonist therapy may effectively treat a resultant tumor, forexample, by stopping the growth of, shrinking in size, and/oreradicating the resultant tumor. Means for measuring the efficacy ofsuch treatment on spreading and metastatic cancers are disclosed hereinelsewhere.

By improvement of cervical/vaginal cancer and dysplasia symptoms, wemean reduction in the size or complete resolution of precancerous and/orcancerous lesions including primary cancers as well as metastaticdisease, absence of progression of precancerous lesions (see also part Dbelow), absence of recurrence, and/or increased time of survival.

C. Estrogen Antagonist Therapy

The present invention is suitable for estrogen antagonist therapy. By“estrogen antagonist,” we mean a composition that will act as aninhibitor of Estrogen Receptor alpha (ERα).

In one embodiment, a complete estrogen antagonist such as fulvestrant isused. By “complete estrogen antagonists,” we mean estrogen receptorantagonists that act as an inhibitor to ERα and have no agonist effectsin any tissue.

In other embodiments, the antagonist may be selected from the listbelow. (One may wish to consult Shelly, et al., CME Review article, Vol.63, No. 3, Obstetrical and Gynecological Survey, 2008, incorporated byreference herein for lists of estrogen antagonists). These include SERMs(Selective Estrogen Receptor Modulators) that have both agonist andantagonist effects, depending on the tissue of interest. PreferableSERMs would include those that have antagonistic effects in thecervix/vagina.

DRUGS TARGETING ESTROGEN-ER PATHWAY Effect in cervix, vagina and DrugStatus endometrium* SERMs Fulvestrant approved antagonist in cervix,vagina, and endometrium Ormeloxifene approved antagonist in endometriumRaloxifene approved neutral in endometrium Tamoxifen approved agonist incervix, vagina, and endometrium Toremifene approved agonist inendometrium Arzoxifene in clinical trials antagonist in endometriumBazedoxifene approved in Europe only antagonist in endometriumPipendoxifene in clinical trials antagonist in endometrium Lasofoxifeneapproved in Europe only neutral in endometrium HMR-3339 in clinicaltrials undefined Ospemifene in clinical trials agonist in vagina andendometrium Acolbifene (EM-652) in clinical trials antagonist inendometrium EM-800 clinical development antagonist in endometriumterminated Droloxifene clinical development agonist in endometriumterminated Idoxifene clinical development agonist in endometriumterminated Levormeloxifene clinical development agonist in endometriumterminated *Undefined if not mentioned. Also note that the effect ofmost drugs on cervix is not defined.

D. Prevention of Cervical/Vaginal Cancers

The present invention is also useful for the prevention ofcervical/vaginal cancers. This is especially appropriate in situationswhere a patient has been diagnosed with pre-cancerous lesions or hasbeen treated to remove cancers but is at risk of recurrence. Treatmentvia the estrogen antagonist therapies of the present invention mayobviate the need for surgical intervention.

We envision that use of estrogen antagonist therapies of the presentinvention to prevent initial or recurrent cervical/vaginal cancers willbe similar to the treatment protocols. Once an appropriate patient hasbeen identified, the patient will be treated with an effective amount ofthe anti-estrogen therapy such that there is no progression of thedisease to cervical cancer. In another embodiment, the patient istreated and there is a slowing of the progression of the disease.

EXAMPLES Example 1 Treatment of Cervical/Vaginal Cancers andPrecancerous Cervical/Vaginal Lesions

The majority of human cervical cancers are associated with the high-riskhuman papillomaviruses (HPVs), which encode the potent E6 and E7oncogenes. Using a mouse model for HPV-associated cancers in the lowerfemale reproductive tract, we have previously demonstrated that estrogenis required for genesis of cervical and vaginal cancers. These data areconsistent with the observation that long-term use of oralcontraceptives or multiple pregnancies significantly increases the riskfor cervical cancer in HPV-infected women. Although ERα is required forthe development of atypical squamous metaplasias, which is proposed tobe the first step of cervical carcinogenesis preceding the developmentof CIN, it is unclear whether ERα is crucial for the development andmaintenance of cervical/vaginal cancers.

In the present study, we examined whether an estrogen antagonist iseffective in treating cervical/vaginal cancer and dysplasia arising inK14E6/K14E7 double transgenic mice treated with exogenous estrogen for 6months. We experimentally demonstrated for the first time that acomplete anti-estrogen, fulvestrant, efficiently clears cancers in thelower reproductive tracts of female mice. Also important is thatfulvestrant treatment caused the complete regression of dysplasticlesions. Based on these findings, we propose the use of anti-estrogensin controlling gynecological disease in the lower reproductive tracts inwomen.

Materials and Methods

Mice. K14E7 and K14E6 transgenic mice were described previously (Herberet al., 1996; Song et al., 1999). Experimental mice were generated bycrossing K14E7 hemizygote and K14E6 homozygote mice. Female progenieswere genotyped by PCR and a slow-releasing 17β-estradiol (E₂) tablet(0.05 mg/60 days) (Innovative Research of America) was insertedsubcutaneously under the dorsal skin every two months beginning at 4-6weeks of age. After 6-month treatment with E₂, one group of mice wassacrificed immediately to evaluate cancer phenotypes (6M E₂ group),another group was maintained for another month without further treatment(6M E₂+1M without fulvestrant group), and the third group was treatedwith fulvestrant for a month (6M E₂+1M with fulvestrant group). Micewere injected intraperitoneally with 0.3 ml of bromo-deoxyuridine (BrdU)(12.5 mg/ml) 1 hr prior to euthanasia to measure cellular proliferation.Female reproductive tracts were harvested and processed as previouslydescribed (Riley et al., 2003). Mice were housed in McArdle LaboratoryAnimal Care Unit of the University of Wisconsin Medical School approvedby the Association for Assessment of Laboratory Animal Care. Allprocedures were carried out according to an animal protocol approved bythe University of Wisconsin Medical School Institutional Animal Care andUse Committee.

Fulvestrant treatment. Faslodex (50 mg/ml, human formulation ofintramuscular injection of fulvestrant) was purchased from Astra Zeneca.Mice were subcutaneously injected with 0.15 ml (equivalent to 7.5 mg offulvestrant) of Faslodex twice a week for a month (total of 9injections).

Hematoxylin and eosin staining. Hematoxylin and eosin (H&E) staining wasperformed as previously described (Riley et al., 2003).

Statistical analyses were performed using one of a Log rank test, atwo-sided Fisher's exact test, or a Wilcoxon rank sum test.

Results & Discussions

Cervical cancer incidence in K14E6/K14E7 double transgenic mice treatedwith estrogen for 6 months and then with fulvestrant for another month.To evaluate potential use of SERMs as therapeutics for cervical cancer,we have chosen to use fulvestrant, a complete anti-estrogen approved forclinical uses, because fulvestrant is well known to inhibit ERα in allexamined mouse and human tissues, unlike tamoxifen and toremifene(Shelly et al., 2008). We first tested two doses of fulvestrant (10mg/week and 15 mg/week) to determine an effective dose that reducesuterine wet weight after treatment for a week. The indicated dose wasgiven to nontransgenic female mice by two injections at day 0 and day 3,and uteri were harvested and weighed at day 7. Uterine wet weight wasnormalized to body weight.

The dose range used was based upon the prior literature (Ariazi et al.,2006). Although 10 mg decreased uterine wet weight by 50%, 15 mg causedfurther reduction in uterine wet weight by 70% (data not shown). Wechose 15 mg/week of fulvestrant to ensure maximum effect of fulvestranton female reproductive tracts. We also used K14E6/K14E7 doubletransgenic mice to ensure high incidence of cervical cancer and moreclosely mimic clinical settings in which both E6 and E7 genes are alwayspresent (Brake and Lambert, 2005; zur Hausen, 2002). Mice were treatedwith exogenous E₂ (using slow release pellets that deliver 0.05 mg/60days, a physiological dose sufficient to induce continuous estrus) for 6months, a treatment period sufficient for the development of cervicalcancers in 95% of K14E6/K14E7 double transgenic mice (Riley et al.,2003). Mice were subsequently injected with fulvestrant as described inMaterials and Methods. Reproductive tracts were harvested, fixed inpara-formaldehyde (PFA), and embedded in paraffin. Every tenth 5 μMsection was stained with H&E and histopathologically scored to identifythe worst grade of cervical/vaginal disease present in each animal.Consistent with previous results (Riley et al., 2003), all ofK14E6/K14E7 mice treated for six months with estrogen (6M E₂ group)developed cancers in the lower reproductive tracts (cervix and vagina)(Tables 1 and 2). All mice that were treated with estrogen for 6 monthsand maintained for another month without further treatment (6M E₂+1Mwithout fulvestrant group) also developed lower reproductive tractcancers at a level similar to those of the 6M E₂ group (Tables 1 and 2).Strikingly, mice that were treated with estrogen for 6 months and thensubsequently treated with fulvestrant for one month (6M E₂+1M withfulvestrant group) displayed dramatically reduced cancer incidence (15%)(p=0.0001, compared to 6M E₂+1M without fulvestrant group, two-sidedFisher's exact test).

Comparison of cancers in K14E6/K14E7 double transgenic mice in threedifferent treatment groups. To determine whether withdrawal of exogenousestrogen for a month results in partial regression of cervical cancer,we compared cancers arising in 6M E₂ group and 6M E₂+1M withoutfulvestrant group (Table 2). These two groups showed comparable cancerincidence, average size of the largest tumors (p>0.5), and cancermultiplicity (p=0.09). However, treatment with fulvestrant for one monthcaused highly significant reduction in all three cancer-characteristicscompared to both 6M E₂ and 6M E₂+1M without fulvestrant groups (p<0.003,two-sided Fisher's exact test or Wilcoxon rank sum test) (Table 2). Wehave previously shown that withdrawal of exogenous estrogen for threemonths results only in partial regression of cervical cancers, which ismuch less dramatic than fulvestrant treatment (p=0.009, two-sidedFisher's exact test). Taken together, we conclude that endogenousestrogen plays a role in maintaining cervical cancer in K14E6/K14E7double transgenic mice.

Comparison of vaginal disease in K14E6/K14E7 double transgenic mice inthree different treatment groups. Because too small a number of vaginalcancers arose in our experimental animals, it was difficult to comparethree groups of mice described above for cancer characteristics such ascancer incidence, tumor multiplicity, and cancer size. Instead, wecompared overall vaginal disease states in those mice by givingarbitrary scores for each disease state as described in Table 1. It wasnotable that the treatment with fulvestrant led to highly significantreduction in severity of vaginal disease compared to that in non-treatedmice (p=0.0008, two-sided Wilcoxon rank sum test) (Table 1), whereas the6M E2 group and the 6M E2+1M without fulvestrant group displayed asimilar degree of vaginal disease (p=0.6). It was also noted thatcervical disease is more severe than vaginal disease (p=0.0004,two-sided Wilcoxon rank sum test).

Potential implication of cancers persisting after fulvestrant treatment.It was immediately noted that epithelia of lower reproductive tractsfrom fulvestrant-treated mice are thin with only 2-3 layers of cells,indicating that ERα function is crucial for thickening ofcervical/vaginal epithelium (FIG. 1). This result is in agreement withthe observation that lower reproductive tract epithelium is hypoplasticin ERα-deficient tissue recombinant (Buchanan et al., 1998) and mice(Chung et al., 2008). Unclear is the nature of glassiness in cervicalepithelium of fulvestrant-treated mice and glassiness increases astreated with fulvestrant for longer periods of time (FIG. 1). Glassyepithelia were less prominent in the vagina compared to the cervix (datanot shown). It was particularly interesting to note that epithelia nearthe cancers remaining after 1M-fulvestrant treatment were as thick asthose in mice not treated with fulvestrant (FIG. 2), indicating thatfulvestrant is not so effective in those specific areas as in otherregions in these mice. One possibility is that in these cases, thecancers and/or the associated stroma are producing their own estrogen,which leads to locally elevated estrogen concentrations that aresufficient to suppress fulvestrant's antagonistic function locally. Ifthis prediction is correct, then aromatase inhibitors in conjunctionwith fulvestrant may potentially be more effective in controlling suchlower reproductive tract cancers.

Fulvestrant causes regression of dysplastic lesions. In thehistopathological analyses of lower reproductive tracts, we alsoobserved the absence of any grade of dysplastic lesions (CIN and VIN) inmice treated with estrogen for 6 months and then administeredfulvestrant for 1 month, whereas dysplasias were found in the entirelower reproductive tracts of mice treated with estrogen but not withfulvestrant (FIG. 1B). Dysplastic lesions were also absent in micetreated with estrogen for 6 months and then with fulvestrant for twoweeks even though these animals still had cancers in the lowerreproductive tracts (FIG. 1). Furthermore, only focal dysplastic lesionswere observed in the epithelia of mice still bearing cancers afterfulvestrant treatment for a month (FIG. 2). These results indicate thatfulvestrant is effective in treating not only cervical/vaginal cancersbut also dysplastic lesions in the female lower reproductive tracts ofmice.

Potential use of anti-estrogen therapy for lower reproductive tractdisease in women. Results of our current study strongly suggest thatanti-estrogen therapy could be effective for treating and/or preventinglower reproductive tract disease including cancers in women. There is noindication that fulvestrant, the anti-estrogen used in this study, hasestrogenic effect in women. However, this complete anti-estrogenicproperty may not be necessarily beneficial for long-term therapy becauseabsence of estrogen function may cause other clinical symptoms seen inpostmenopausal women. In this regard, raloxifene might be a betteroption because it shows protective effect on bone and cardiovascularsystem, whereas it has anti-estrogenic activity in breast and uterinetissues (Shelly et al., 2008). Therefore, we carried out additionalstudies and found that raloxifene also can eliminate pre-existingcervical diseases including cervical cancer (See Example 2, below). Ifestrogen is necessary for lower reproductive tract disease in women,aromatase inhibitors are anticipated to be as effective asanti-estrogens because they will also inhibit ER by inhibiting synthesisof its ligand estrogen. This is potentially relevant to our observationthat some mice that were treated with estrogen for 6 months and thenfulvestrant for another month still had cancers with adjacent epitheliumwith normal thickness (FIG. 2). In this context, it is known that somehuman cervical cancers synthesize estrogen by over-expressing aromatase,a key enzyme in estrogen biosynthesis (Nair et al., 2005).

TABLE 1 State of Lower Reproductive Tract Disease¹. Total DysplasiaCervical number No CIN1 CIN2 CIN3 (vaginal) Treatment² of mice dysplasia(VIN1) (VIN2) (VIN3) cancer 6M E₂ 6 0 0 (0) 0 (1) 0 (2) 6 (3) 6M E₂ + 1Mwithout fulvestrant 14 0 0 (0) 0 (3) 3 (6) 11 (5)  6M E₂ + 1M withfulvestrant 13 11 0 (0) 0 (0) 0 (0) 1 (1) ¹Mice were scoredhistopathologically for the worst state of disease present in the cervixor, in parentheses, the vagina. The numbers of mice with the indicatedstate of disease are indicated in each column. ²Female reproductivetracts were harvested immediately after 6-month treatment of estrogen(first row). Mice were treated with estrogen for 6 months and thenfurther kept for another month without (second row) or with (third row)fulvestrant treatment. CIN, cervical intraepithelial neoplasia; VIN,vaginal intraepithelial neoplasia. Note: for Wilcoxon rank sum test (seetext), CIN (VIN) lesions were given the following arbitrary score; nodysplasia = 1; CIN1 (VIN1) = 2; CIN2 (VIN2) = 3; CIN3 (VIN3) = 4; cancer= 5.

TABLE 2 Comparison of cancers in estrogen-treated K14E6/K14E7 mice witha different scheme of further treatment with fulvestrant. Cancer Averagesize Tumor incidence of the largest multiplicity Treatment¹ (%) tumor(mm²) Cervix Vagina 6M E₂ 100² 0.44 ± 0.57² 3.3 ± 1.1^(2,3) 0.5 ± 0.5 6ME₂ + 1M  93² 0.28 ± 0.29² 2.3 ± 1.4^(2,3) 0.4 ± 0.6 without fulvestrant6M E₂ + 1M  15 0.02 ± 0.05  0.2 ± 0.5  0.1 ± 0.3 with fulvestrant¹Treatment groups are identical to those described in Table 1. ²Comparedwith 6M E2 + 1M with fulvestrant, p < 0.008, two-sided Fisher's exacttest (cancer incidence) or Wilcoxon rank sum test (tumor sizes andmultiplicities). ³Compared with vaginal cancer multiplicity, p < 0.008,two-sided Wilcoxon rank sum test.

Example 2 Raloxifene Promotes Regression of Cervical/Vaginal DiseaseIncluding Cancers

EVISTA® tablets were purchased from Eli Lilly and Company, which arehuman formulation of raloxifene (60 mg/tablet). Tablets were resuspendedin phosphate buffered saline at a final concentration of 10 mg/ml.K14E6/K14E7 double transgenic mice were treated with E2 for 6 months andsubsequently with raloxifene for a month by intraperitoneally injectingthem with 0.15 ml of raloxifene suspension (equivalent to 1.5 mg) 5 daysa week. A preferred human dosage is 60 mg daily. As shown in Table 3,raloxifene treatment induced complete regression of cervical and vaginaldisease including cancers.

TABLE 3 Comparison of cervial/vaginal disease in E2-treated K14E6/K14E7mice with or without raloxifene (Ral) treatment. Worst disease state #mice w/ # mice with no dysplasia only # mice w/cancer Treatment groupdysplasia or cancer CIN1 CIN2 CIN3 and dysplasia (%) (group size) CervixVagina (VIN1) (VIN2) (VIN3) Cervix Vagina 6M E2 + 1M without Ral 0 0 0(0) 0 (3) 3 (6) 11 (79) 5 (36) 6M E2 + 1M with Ral 7 6 0 (0) 0 (0) 0 (1)0 (0) 0 (0)  CIN, cervical intraepithelial neoplasia; VIN, vaginalintraepithelial neoplasia. Note: for Wilcoxon rank sum test, arbitraryscores were given to each lesion as following; no dysplasia = 1; CIN1(VIN1) = 2; CIN2 (VIN2) = 3; CIN3 (VIN3) = 4; cancer = 5. Two-sidedstatistical analyses on two groups (6M E2 + 1M without Ral vs. 6M E2 +1M with Ral), cervical cancer incidence p = 0.001 (Fisher's exact test);cervical disease p = 0.00005 (Wilcoxon rank sum test); vaginal cancerincidence p = 0.12 (Fisher's exact test); vaginal disease p = 0.0009(Wilcoxon rank sum test).

Example 3 Fulvestrant Treatment Prevents the Development ofCervical/Vaginal Cancers

CIN and VIN are thought to be the precursor lesions to cervical andvaginal cancers, respectively. Because these lesions largely disappearedin the 6M E2+1M with fulvestrant and 6M E2+1M with Ral groups, weperformed an additional experiment to determine whether fulvestrant canprevent the onset of cervical/vaginal cancers.

To address this issue we began treating mice with fulvestrant after onlythree months of E2 treatment, a time period at which CIN/VIN are presentbut no cancers have yet developed (Table 4, 3M E2 group). Mice treatedwith fulvestrant for one month at the three-month time point (6M E2+preventative fulvestrant group) showed neither CIN nor cervical cancerat the six-month endpoint. These phenotypes are significantly differentfrom those of the 6M E2 group that had not received fulvestrant(p=0.002, cancer incidence; p=0.0009, cervical disease). Thus,fulvestrant not only promotes regression of cervical cancer but alsoprevents the development of this malignancy in mice. The 6M E2+preventative fulvestrant group also displayed a significantly lowerdegree of vaginal disease compared to the 6M E2 group (p=0.002). Thesedata indicate that treatment with an anti-estrogen can preventprogression of precancerous lesions. This, together with our datashowing a therapeutic effect on treating existing cancer also lead tothe prediction that anti-estrogens could be useful in preventingrecurrence of cervical/vaginal cancers in women.

TABLE 4 Comparison of cervical/vaginal disease in E2-treated K14E6/K14E7mice with or without fulvestrant treatment. Worst disease state # micew/ # mice with no dysplasia only # mice w/cancer Treatment groupdysplasia or cancer CIN1 CIN2 CIN3 and dysplasia (%) (group size) CervixVagina (VIN1) (VIN2) (VIN3) Cervix Vagina 3M E2 (n = 6) 0 2 1 (2) 3 (2)2 (0) 0 (0) 0 (0) 6M E2 + preventive fulvestrant 6 6 0 (0) 0 (0) 0 (0) 0(0) 0 (0) 6M E2 (n = 6) 0 0 0 (0) 0 (1) 0 (2)  6 (100)  3 (50)

Example 4 Estrogen Facilitates Tumor Formation by a Human CervicalCancer Cell Line in Nude Mice and Fulvestrant Inhibits Tumor GrowthMethods

Cell lines CVX1 (Bellone et al., 2003) and CIN612E (Bedell et al., 1991)cells were maintained in a mixture of DMEM and F12 (3:1) supplementedwith 5% fetal bovine serum (FBS), 0.4 μg/ml hydrocortisone, 5 μg/mlinsulin, 0.1 nM cholera toxin, 24.2 μg/ml adenine, 10 ng/ml epidermalgrowth factor, 10 Unit/ml penicillin and 10 μg/ml streptomycin. Theother cells (see FIG. 3) were grown in DMEM containing 10% FBS andpenicillin/streptomycin (All cell culture media are available fromGIBCO® Invitrogen Corp., Carlsbad, Calif.).

Immunoblot Cells were lysed in RIPA buffer (50 mM Tris-Cl, pH 8.0, 150mM NaCl, 1% NP-40 detergent, 0.5% sodium deoxycholate, 0.1% SDS)supplemented with proteinase inhibitors and phosphatase inhibitors.Protein concentration was determined by Bradford method and equal amountof lysates were resolved on 10% SDS-polyacrylamide gel. Proteins weretransferred to a PVDF membrane and blocked with 5% nonfat milk in TBS-T(50 mM Tris-Cl, pH 7.5, 200 mM NaCI, 0.2% Tween-20). The membrane wasincubated with either anti-ERα (Santa Cruz Biotechnology) or anti-actinantibody (Santa Cruz Biotechnology) diluted in TBS-T containing 0.5%nonfat milk (1:1000 dilution for anti-ERα; 1:2500 for anti-actin), andthen with horseradish peroxidase-conjugated goat anti-rabbit IgG(Jackson Immunoresearch Laboratories, Inc., West Grove, Pa.) diluted(1:5000) in TBS-T containing 0.5% nonfat milk. After extensive washes,membranes were developed by enhanced chemilluminescent methods (PierceProtein Research Products, Thermo Fisher Scientific, Rockford, Ill.) asdescribed in the manufacturer's instructions.

Mice treatment Six-week old female nude mice were purchased fromHarlan™. Groups of mice were implanted subcutaneously with estrogenpellets that slowly release 0.05 mg of estrogen over 60 days. Thisregimen results in estrogen concentration in serum just enough to keepanimals in persistent estrus. One week after estrogen treatment, 10⁷cells (CVX1) or 10⁶ cells (CaSki and SiHa) were subcutaneously injectedat the flank. A subset of mice was also subcutaneously injected with 7.5mg of fulvestrant twice a week once tumors formed. Tumor size wasmeasured regularly using a slide caliper.

Results Although it is clear from prior studies in HPV16 transgenic micethat the estrogen-ERα pathway is crucial for cervical cancer in mice(Chung et al., 2008), and ER antagonists are effective in controllingthis malignancy (Chung and Lambert, 2009), it is not known if the sameis true for cervical cancer in women. To address this we pursued studiesmaking use of cell lines derived from human cervical cancers. We firstscreened multiple human cervical cancer cell lines for ERα expression byimmunoblot analysis (FIG. 3). ERα expression was undetectable in widelyused cervical cancer cell lines, CaSki, SiHa, HeLa, and C33A that havebeen grown in laboratory tissue culture for decades. However, we foundthat a relatively newly established, low passage cervical cancer cellline, CVX1, retains expression of ERα. This is consistent withERα-positive status of human cervical cancers in vivo (Chung et al.,2008). Importantly, exogenous estrogen facilitated tumor formation bythis cell line in athymic nude mice (FIG. 4) (p=0.001, Log rank test).Whereas 4 out of 7 mice that were not treated with exogenous estrogendeveloped tumors, all estrogen-treated mice developed tumors (p=0.05,two-sided Fisher's exact test). In addition, tumor latency in untreatedmice was significantly higher than in estrogen-treated mice (62 days vs.46 days; p=0.003, two-sided Wilcoxon rank sum test). In contrast,exogenous estrogen had no effect on tumorigenic properties ofERα-negative cervical cancer cell lines SiHa and CaSki. Both cell linesformed tumors in all animals (n=4 each group) at 15 days after injectionregardless of estrogen treatment (data not shown).

To assess whether ERα-antagonists can inhibit the tumorigenic growth ofhuman cervical cancer derived cell lines, we took mice that had beeninjected subcutaneously with human cervical cancer cell lines, waitedfor tumors to develop, and then treated them with fulvestrant.Fulvestrant blocked tumor growth of the ERα-positive CVX1 cancer cellline but did not block tumor growth of the ERα-negative CaSki cell line(FIG. 5). Specifically, within the two-week time treatment period, CVX1tumors grew 2.5 fold without fulvestrant-treatment but did not grow atall with the treatment (p=0.02, two-sided Wilcoxon rank sum test). Incontrast, CaSki tumors grew 4.7 or 5.1 fold with or without fulvestranttreatment, respectively (p=0.51). These results provide further proof,consistent with that obtained with our HPV transgenic mouse model, thathuman cervical cancers that express ERα depend on estrogen for theirgrowth and respond favorably to treatment with an ERα-antagonist.

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1. A method for treatment of cervical or vaginal cancer or their associated dysplasia (CINI-III, VINI-III)), comprising the steps of (a) identifying a human cervical or vaginal cancer and/or dysplasia (CIN/VIN) patient, (b) administering an effective amount of an estrogen antagonist therapy to the patient, wherein the amount is effective to reduce cancer and dysplasia symptoms, and (c) observing a reduction of cancer and dysplasia symptoms in the patient.
 2. The method of claim 1 wherein the estrogen antagonist therapy is fulvestrant.
 3. The method of claim 2 wherein the estrogen antagonist therapy is administered at a loading dose of 500 mg (±10%) on day 1 and 14 and monthly dosing of either 250 mg (±10%) or 500 mg (±10%).
 4. The method of claim 1 wherein the estrogen antagonist therapy is a selective estrogen receptor modulator with antagonistic effects in the cervix/vagina.
 5. The method of claim 1 wherein the estrogen antagonist therapy is raloxifene.
 6. The method of claim 1 further comprising the step of administering at least one of an aromatase inhibitor and a chemotherapeutic agent.
 7. The method of claim 6, wherein the chemotherapeutic agent is selected from the group consisting of platinum and taxane.
 8. The method of claim 1 wherein the step of observing comprises measuring the reduction of cancer and dysplasia symptoms in the patient.
 9. A method for preventing recurrence of cervical or vaginal cancers comprising the steps of (a) identifying a human cervical or vaginal cancer patient who has been successfully treated to remove detectable cancer, (b) administering an effective amount of an estrogen antagonist therapy to the patient, and (c) monitoring for persistence of a disease-free state.
 10. The method of claim 9 wherein the estrogen antagonist therapy is fulvestrant.
 11. The method of claim 10 wherein the estrogen antagonist therapy is administered at a loading dose of 500 mg (±10%) on days 1 and 14 and monthly dosing of either 250 mg (±10%) or 500 mg (±10%).
 12. The method of claim 9 wherein the estrogen antagonist therapy is a selective estrogen receptor modulator with antagonistic effects in the cervix.
 13. The method of claim 9 wherein the estrogen antagonist therapy is raloxifene.
 14. The method of claim 9 further comprising the step of administering at least one of an aromatase inhibitor and a chemotherapeutic agent.
 15. A method of treating ERα-dependent spreading or metastatic cancers of cervical or vaginal tissue origin, comprising: (a) identifying a human cancer patient with spreading or metastatic cancer of cervical or vaginal tissue origin; (b) administering an effective amount of an estrogen antagonist therapy to the patient; and (c) observing reduction of cancer symptoms in the patient.
 16. The method of claim 15 further comprising the step of administering at least one of an aromatase inhibitor and a chemotherapeutic agent. 